Chromosome instability (CIN) describes both disruptions of chromosome number such as aneuploidy or misegregation (nCIN) as well as disruptions of chromosome structure, such as chromosome translocations or rearrangements (sCIN). Both forms of CIN are associated with cancer. Progressive loss of genome integrity via CIN contributes to loss of heterozygosity, and can cause or exacerbate the disease. In addition to malignancy, disruptions in chromosome structure or number also contribute to defects in human development. For example, trisomy 21 causing Down syndrome can occur via chromosome mis-segregation (nCIN), but also from chromosome fusion via Robertsonian translocation(sCIN). Thus, the mechanisms that maintain chromosome structure and number are integral to human health at many levels. The centromere is essential for the normal segregation of chromosomes; thus, mutations affecting centromere function contribute to numerical CIN and aneuploidy. However, recent work suggests that the centromere is also vulnerable to chromosome rearrangements, fragmentation, and DNA damage, creating structural CIN. This also contributes to segregation defects. Importantly, the mechanisms that prevent sCIN in the centromere are largely unexplored. This proposal investigates structural instability associated with the centromere, specifically at the highly repetitive outer-repeat elements that are usually assembled into heterochromatin. It employs a tractable genetic system: the fission yeast S. pombe, which is well-established as a model for centromere function in higher cells. The project uses genetics, molecular biology, and novel cell biology methods, including live, single cell analysis and super-resolution microscopy, to examine the mechanisms that protect the centromere and preserve its integrity. The first Aim builds on extensive preliminary data to examine how the combination of replication defects and absence of heterochromatin increase the frequency of rearrangements. The second aim asks how proteins that are linked to centromere heterochromatin function in the DNA damage response either through centromere maintenance or effects on the euchromatin effects. The third Aim uses a novel four-chromosome fission yeast strain to study Robertsonian translocation. This is the first yeast model for this common chromosome rearrangement, and will examine evidence for centromere fusion and mechanisms for segregation and translocation in meiosis and mitosis. Together, these approaches will develop a strong mechanistic model for sCIN in the centromere with direct relevance to human health.